Publications by authors named "Hiroshi Izawa"

9 Publications

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Intravitreal aflibercept protects photoreceptors of mice against excessive light exposure.

J Pharmacol Sci 2018 Aug 4;137(4):407-411. Epub 2018 Aug 4.

Molecular Pharmacology, Department of Biofunctional Evaluation, Gifu Pharmaceutical University, 1-25-4 Daigaku-nishi, Gifu, 501-1196, Japan. Electronic address:

Our previous studies found that an anti-placental growth factor (PlGF) antibody protected the retina in light-induced retinal damage model, a model of non-exudative age-related macular degeneration (AMD). Aflibercept is an inhibitor of vascular endothelial growth factor (VEGF) and PlGF. In present study, we revealed that the intravitreal injection of aflibercept lessens light-induced retinal damage, while anti-VEGF antibody has no effect on the light-exposed retina. Moreover, PlGF disrupted the tight junctions between the human retinal pigment epithelial cells in vitro, and aflibercept blocked the disruption. These data suggest that the aflibercept may be an effective treatment of non-exudative AMD.
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http://dx.doi.org/10.1016/j.jphs.2018.07.010DOI Listing
August 2018

Platelet-Derived Growth Factor-BB Lessens Light-Induced Rod Photoreceptor Damage in Mice.

Invest Ophthalmol Vis Sci 2017 12;58(14):6299-6307

Molecular Pharmacology, Department of Biofunctional Evaluation, Gifu Pharmaceutical University, Gifu, Japan.

Purpose: Platelet-derived growth factor (PDGF)-BB is known to have neuroprotective effects against various neurodegenerative disorders. The purpose of this study was to determine whether PDGF-BB can be neuroprotective against light-induced photoreceptor damage in mice.

Methods: Mice were exposed to 8000-lux luminance for 3 hours to induce phototoxicity. Two hours before light exposure, the experimental mice were injected with PDGF-BB intravitreally, and the control mice were injected with phosphate-buffered saline. The light-exposed PDGF-BB-injected mice and saline-injected mice were evaluated electroretinographically and histologically. The site and expression levels of PDGFR-β and PDGF-BB were determined by immunostaining and Western blotting, respectively. The effect of PDGF-BB on light-induced cone and rod photoreceptor damage was also evaluated in vitro in 661W cells, a murine cone photoreceptor cell line, and in primary retinal cell cultures.

Results: An intravitreal injection of PDGF-BB significantly reduced the decrease in the amplitudes of the electroretinograms (ERGs) and the thinning of the outer nuclear layer (ONL) induced by the light exposure. It also reduced the number of TUNEL-positive cells in the ONL. PDGFR-β was expressed in the rod outer segments (OSs) but not the cone OSs. The levels of PDGF-BB and PDGFR-β were decreased after light irradiation. In addition, PDGF-BB had protective effects against light-induced damage to cells of rod photoreceptors but had no effect on the 661W cells in vitro.

Conclusions: These findings indicate that PDGF-BB reduces the degree of light-induced retinal damage by activating PDGFR-β in rod photoreceptors. These findings suggest that PDGF-BB could play a role in the prevention of degeneration in eyes susceptible to phototoxicity.
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http://dx.doi.org/10.1167/iovs.17-22556DOI Listing
December 2017

Both Autocrine Signaling and Paracrine Signaling of HB-EGF Enhance Ocular Neovascularization.

Arterioscler Thromb Vasc Biol 2018 01 30;38(1):174-185. Epub 2017 Nov 30.

From the Molecular Pharmacology, Department of Biofunctional Evaluation, Gifu Pharmaceutical University, Japan (Y.I., M.S., S.N., S.T., Y.H., H.I., T.M., K.T., H.H.); Proteo-Science Center, Division of Cell Growth and Tumor Regulation, Ehime University Shitsukawa, Toon, Japan (T.S., H.N., S.H.); and Department of Biochemistry and Molecular Genetics, Ehime University Graduate School of Medicine, Shitsukawa, Toon, Japan (T.S., S.H.).

Objective: The incidence of blindness is increasing because of the increase in abnormal ocular neovascularization. Anti-VEGF (vascular endothelial growth factor) therapies have led to good results, although they are not a cure for the blindness. The purpose of this study was to determine what role HB-EGF (heparin-binding epidermal growth factor-like growth factor) plays in ocular angiogenesis.

Approach And Results: We examined the role played by HB-EGF in ocular neovascularization in 2 animal models of neovascularization: laser-induced choroidal neovascularization (CNV) and oxygen-induced retinopathy. We also studied human retinal microvascular endothelial cells in culture. Our results showed that the neovascularization was decreased in both the CNV and oxygen-induced retinopathy models in HB-EGF conditional knockout mice compared with that in wild-type mice. Moreover, the expressions of HB-EGF and VEGF were increased after laser-induced CNV and oxygen-induced retinopathy, and their expression sites were located around the neovascular areas. Exposure of human retinal microvascular endothelial cells to HB-EGF and VEGF increased their proliferation and migration, and CRM-197 (cross-reactive material-197), an HB-EGF inhibitor, decreased the HB-EGF-induced and VEGF-induced cell proliferation and migration. VEGF increased the expression of mRNA. VEGF-dependent activation of EGFR (epidermal growth factor receptor)/ERK1/2 (extracellular signal-regulated kinase 1/2) signaling and cell proliferation of endothelial cells required stimulation of the ADAM17 (a disintegrin and metalloprotease) and ADAM12. CRM-197 decreased the grades of the fluorescein angiograms and size of the CNV areas in marmoset monkeys.

Conclusions: These findings suggest that HB-EGF plays an important role in the development of CNV. Therefore, further investigations of HB-EGF are needed as a potential therapeutic target in the treatment of exudative age-related macular degeneration.
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http://dx.doi.org/10.1161/ATVBAHA.117.310337DOI Listing
January 2018

Toll-like receptor 4 inhibitor protects against retinal ganglion cell damage induced by optic nerve crush in mice.

J Pharmacol Sci 2017 Mar 2;133(3):176-183. Epub 2017 Mar 2.

Department of Biofunctional Evaluation, Molecular Pharmacology, Gifu Pharmaceutical University, 1-25-4 Daigaku-nishi, Gifu 501-1196, Japan. Electronic address:

Toll-like receptor 4 (TLR4) plays key roles in innate immune responses and inflammatory reactions. TAK-242 (resatorvid) is a small-molecule cyclohexene derivative that selectively inhibits TLR4 signaling pathways and suppresses inflammatory reactions. Here we investigated the protective effects of TAK-242 against optic nerve crush (ONC) which induces axonal injury like glaucoma in mice. TAK-242 was injected intravitreally immediately after ONC. The effect of TAK-242 was evaluated by measuring the number of fluorogold-labeled retinal ganglion cells (RGCs) at 10 days after ONC. Furthermore, the expression levels of phosphorylated-nuclear factor-kappa B (p-NF-κB) and phosphorylated-p38 (p-p38) were measured by Western blotting. In addition, we examined activated astrocytes by immunostaining. TAK-242 significantly abrogated the loss of RGCs associated with ONC. Moreover, the expression levels of p-NF-κB and p-p38 were significantly reduced by TAK-242 treatment. Furthermore, TAK-242 and C34, a TLR4 inhibitor, significantly reduced astrocyte activation in the ganglion cell and inner plexiform layers, compared with vehicle treatment. These findings indicate that TAK-242 inhibits not only the TLR4 signaling pathway but also astrocyte activation downstream of this pathway, suggesting that the inhibition of TLR4 signaling is a promising candidate for the treatment of glaucoma.
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http://dx.doi.org/10.1016/j.jphs.2017.02.012DOI Listing
March 2017

Astaxanthin Protects Against Retinal Damage: Evidence from In Vivo and In Vitro Retinal Ischemia and Reperfusion Models.

Curr Eye Res 2016 11 9;41(11):1465-1472. Epub 2016 May 9.

a Molecular Pharmacology, Department of Biofunctional Evaluation , Gifu Pharmaceutical University , Gifu , Japan.

Purpose: Astaxanthin exhibits various pharmacological activities, including anti-oxidative, anti-tumor, and anti-inflammatory effects, and is thought to exert a neuroprotective effect via these mechanisms. The purpose of this study was to investigate the protective effects of astaxanthin on neuronal cell death using a retinal ischemia/reperfusion model.

Methods: In vivo, retinal ischemia was induced by 5 h unilateral ligation of the pterygopalatine artery (PPA) and the external carotid artery (ECA) in ddY mice. Astaxanthin (100 mg/kg) was administered orally 1 h before induction of ischemia, immediately after reperfusion, at 6 or 12 h after reperfusion, and twice daily for the following 4 days. Histological analysis and an electroretinogram (ERG) were performed 5 days after ischemia/reperfusion. In vitro, cell death was induced in the RGC-5 (retinal precursor cells) by oxygen-glucose deprivation (OGD), and the rates of cell death and production of intracellular reactive oxygen species (ROS) were measured using nuclear staining and a ROS reactive reagent, CM-HDCFDA.

Results: Histological studies revealed that astaxanthin significantly reduced retinal ischemic damage and ERG reduction. In in vitro studies, astaxanthin inhibited cell death and ROS production in a concentration-dependent manner.

Conclusions: Collectively, these results indicate that astaxanthin inhibits ischemia-induced retinal cell death via its antioxidant effect. Hence, astaxanthin might be effective in treating retinal ischemic pathologies.
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http://dx.doi.org/10.3109/02713683.2015.1127392DOI Listing
November 2016

Protective effects of NSP-116, a novel imidazolyl aniline derivative, against light-induced retinal damage in vitro and in vivo.

Free Radic Biol Med 2016 07 2;96:304-12. Epub 2016 May 2.

Molecular Pharmacology, Department of Biofunctional Evaluation, Gifu Pharmaceutical University, Japan. Electronic address:

In this study, we investigated the protective effects of NSP-116 [4-(4-acetylpiperazin-1-yl)-2-(1H-imidazol-1-yl) aniline], a novel imidazolyl aniline derivative, against light-induced photoreceptor cell damage. In an in vitro experiment, murine photoreceptor (661W) cells were damaged by exposure to light for 24h. Viability of 661W cells after light exposure was assessed by Hoechst 33342/Propidium iodide nuclear staining and a tetrazolium salt (WST-8) assay. Intracellular radical production in 661W cells was evaluated using the reactive oxygen species (ROS) sensitive probe 5-(and 6)-chloromethyl-2, 7-dichlorodihydrofluorescein diacetate acetyl ester (CM-H2DCFDA). NSP-116 significantly suppressed light-induced cell death and ROS production in 661W cells. In an in vivo mouse experiment, retinal damage was induced by exposure to white light at 8000lx for 3h after dark adaptation. Retinal damage was evaluated by recording the electroretinogram and measuring the outer nuclear layer (ONL) thickness at 5 days after light exposure. Single oral administration of NSP-116 before light exposure protected retinal function and ONL thinning after light exposure. Furthermore, the effect of NSP-116 on lipid peroxidation was evaluated using thiobarbituric acid reactive substance (TBARS) assay in porcine retina, and was found to decrease the production of TBARS. Electron spin resonance (ESR) measurements showed that NSP-116 exhibited radical scavenging activities against 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, superoxide anion radical (∙O2(-)), and hydroxyl radical (∙OH). These findings suggest that NSP-116 has protective effects against light-induced photoreceptor degeneration in vitro and in vivo as a free radical scavenger, and it may be a novel therapeutic agent for retinal degenerative disorders, such as dry age-related macular degeneration (AMD).
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http://dx.doi.org/10.1016/j.freeradbiomed.2016.03.036DOI Listing
July 2016

Progranulin promotes the retinal precursor cell proliferation and the photoreceptor differentiation in the mouse retina.

Sci Rep 2016 Mar 31;6:23811. Epub 2016 Mar 31.

Molecular Pharmacology, Department of Biofunctional Evaluation, Gifu Pharmaceutical University, 1-25-4 Daigaku-nishi, Gifu 501-1196, Japan.

Progranulin (PGRN) is a secreted growth factor associated with embryo development, tissue repair, and inflammation. In a previous study, we showed that adipose-derived stem cell-conditioned medium (ASC-CM) is rich in PGRN. In the present study, we investigated whether PGRN is associated with retinal regeneration in the mammalian retina. We evaluated the effect of ASC-CM using the N-methyl-N-nitrosourea-induced retinal damage model in mice. ASC-CM promoted the differentiation of photoreceptor cells following retinal damage. PGRN increased the number of BrdU(+) cells in the outer nuclear layer following retinal damage some of which were Rx (retinal precursor cell marker) positive. PGRN also increased the number of rhodopsin(+) photoreceptor cells in primary retinal cell cultures. SU11274, a hepatocyte growth factor (HGF) receptor inhibitor, attenuated the increase. These findings suggest that PGRN may affect the differentiation of retinal precursor cells to photoreceptor cells through the HGF receptor signaling pathway.
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http://dx.doi.org/10.1038/srep23811DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4814875PMC
March 2016

Protective Effects of Antiplacental Growth Factor Antibody Against Light-Induced Retinal Damage in Mice.

Invest Ophthalmol Vis Sci 2015 Oct;56(11):6914-24

Purpose: Placental growth factor (PlGF) is part of the VEGF family and is known to be involved in angiogenesis, vasopermeability, and neuroprotection. Recently, PlGF has been reported as a novel therapeutic target for wet AMD. However, there are few reports about the effect of PlGF against dry AMD. Previously, we reported that PlGF has protective effects against retinal neuronal cell damage in vitro. Therefore, we investigated the effects of PlGF against photoreceptor degeneration.

Methods: In this study, mice were exposed to white light at 8000 lx for 3 hours to induce retinal damage, which was evaluated by recording the electroretinogram amplitude and measuring the outer nuclear layer (ONL) thickness. The mice were injected intravitreally with PlGF before light exposure, PlGF after light exposure, or anti-PlGF antibody before light exposure. RPE-choroid-sclera flat mounts were immunostained with anti-ZO-1 antibody to evaluate the disruption of retinal pigmented epithelium (RPE) cell-cell junctional integrity after light exposure. Furthermore, the expression of VEGF receptor in the retina and RPE-choroid complex after light exposure was measured using Western blot analysis.

Results: Contrary to the expected outcome, PlGF treatment exacerbated the light-induced retinal functional damage and ONL thinning. In contrast, anti-PlGF treatment significantly improved the light-induced retinal degeneration. The disruption of RPE cell-cell junctional integrity after light exposure was suppressed by anti-PlGF treatment. Moreover, the VEGF receptor, which is involved in blood-retinal barrier breakdown, was up-regulated after light exposure.

Conclusions: These findings suggest that anti-PlGF antibody has protective effects against light-induced retinal degeneration in the murine retina through inhibition of RPE breakdown after light exposure. Thus, anti-PlGF antibody may be useful therapeutic agents in dry AMD.
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http://dx.doi.org/10.1167/iovs.15-16748DOI Listing
October 2015

Involvement of endoplasmic reticulum stress in optic nerve degeneration after chronic high intraocular pressure in DBA/2J mice.

J Neurosci Res 2015 Nov 13;93(11):1675-83. Epub 2015 Aug 13.

Molecular Pharmacology, Department of Biofunctional Evaluation, Gifu Pharmaceutical University, Gifu, Japan.

DBA/2J mice are one of several animal strains used for experimental models of both intraocular hypertension and glaucoma. This study investigates the relationship between endoplasmic reticulum (ER) stress and optic nerve degeneration in DBA/2J mice. Intraocular pressure (IOP) was measured in DBA/2J mice between the ages of 6 and 15 months. Optic nerve damage was assessed at 15 months of age. The nerve was immunostained with antibodies to either neurofilament heavy chain (NFH) or phosphorylated NFH (pNFH), and optic nerve damage was assessed by performing NFH- and pNFH-positive axon counts. Expression levels of the ER stress proteins 78-kDa glucose-regulated protein, also known as binding immunoglobulin protein, and C/EBP homologous protein were assayed with Western blotting. We also investigated ER stress localization in the optic nerve by double immunostaining with antibodies to ionized calcium-binding adaptor molecule 1, myelin basic protein, and glial fibrillary acidic protein (GFAP). In DBA/2J mice, IOP began to rise at 8 months of age, and retinal degeneration was detected at 15 months of age. DBA/2J mice had fewer axons than controls at 15 months of age. ER stress-related protein levels were higher in the optic nerves of DBA/2J mice and were colocalized with GFAP-positive astrocytes. Our findings suggest that ER stress plays a role in optic nerve degeneration during chronic ocular hypertension. Furthermore, ER stress may be related in some way to astrocyte activation.
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http://dx.doi.org/10.1002/jnr.23630DOI Listing
November 2015
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